The Role of Genetic Variation in the Fungal/Host Interaction

描述（由申请人提供）：本提案的目标是破译真菌被先天免疫系统识别的机制以及真菌逃避识别的机制。病原真菌如白色念珠菌和非病原体酿酒酵母通过遗传和结构变异来伪装其表面免疫决定因子，使其免受巨噬细胞和中性粒细胞的攻击。最近对白色念珠菌基因组序列的分析表明，它编码RNAi系统的关键蛋白质（在酵母菌中缺失），这可能是遗传变异的关键因素。序列分析显示，白色念珠菌具有Argonaute和Dicer的同源物，这是抑制RNA途径的两个关键成分，在其他系统中已知可以抑制移动遗传元件的转位。此外，它还产生与LINE-1反转录转座子Zorro-3同源的小rna，可能是干扰rna。敲除假定的Argonaute和Dicer基因将决定这些蛋白质是否构成产生这些rna的途径的要素。这些突变体与标记的Zorro-3元件将允许确定该系统的丢失是否增强转位，以及该RNAi系统是否影响念珠菌的形态发生和毒力。体外和体内实验旨在鉴定识别念珠菌的先天免疫系统的组成部分。体外筛选利用小鼠RNAi文库来鉴定负责念珠菌吞噬和丝化的巨噬细胞基因。与此相一致，假丝酵母突变体将被构建，在其表面上具有暴露的关键特征分子。这些突变体将与这些基因敲低一起用于重建从受体到细胞因子生产的途径以及这些细胞表面特征的特定受体。体内实验旨在鉴定2-葡聚糖受体Dectin-1在标记短序列“Sortag”的转基因小鼠中识别真菌的功能。这些小鼠从其自身的启动子中表达一个分类标记的Dectin-1，将允许鉴定与Dectin-1相互作用的蛋白质，表达Dectin-1的不同免疫细胞的集合，以及在体内识别真菌识别的功能。sortagged Dectin-1小鼠的可用性将为鉴定抗真菌药物的新靶标提供独特的机会。公共卫生相关性：我们对真菌疾病的防御涉及到免疫系统对入侵病原体的识别。该研究将揭示该检测系统的机制和真菌用来避免识别的策略。为了避免被识别，病原体会改变或掩盖识别特征。了解这种遭遇将指导新抗生素的设计。

DESCRIPTION (provided by applicant): The goal of this proposal is to decipher the mechanism by which fungi are recognized by the innate immune system and the mechanisms used by fungi to evade recognition. Pathogenic fungi such as Candida albicans, and the non-pathogen, S. cerevisiae camouflage their surface immune determinants from macrophages and neutrophils by genetic and structural variation. Recent analysis of the genome sequence of Candida albicans suggests that it encodes the crucial proteins of the RNAi system (missing from Saccharomyces) that could be a key contributor to genetic variation. Sequence analysis reveals that C. albicans has homologues of Argonaute and Dicer, two key components of an inhibitory RNA pathway, known in other systems to suppress the transposition of mobile genetic elements. Moreover, it also produces small RNAs, likely interfering RNAs, which have homology to the LINE-1 retrotransposon, Zorro-3. Knockouts of the putative Argonaute and Dicer genes will determine whether these proteins constitute elements of the pathway to create these RNAs. These mutants together with a tagged Zorro-3 element will permit determination of whether loss of this system enhances transposition and whether this RNAi system affects morphogenesis and virulence of Candida. Both in vitro and in vivo experiments are designed to identify the components of the innate immune system that recognize Candida. An in vitro screen utilizes a murine RNAi library to identify macrophage genes responsible for phagocytosis and filamentation of Candida. In concert with this screen Candida mutants will be constructed that have unmasked key signature molecules on their surface. These mutants will be used together with these knockdowns to reconstruct both the pathways from receptor to cytokine production and the specific receptors for these cell surface signatures. The in vivo experiments are designed to identify the function of the 2-glucan receptor, Dectin-1 in recognizing fungi in transgenic mice tagged with a short sequence, the "Sortag". These mice expressing a sortagged Dectin-1 from its own promoter will permit identification of proteins that interact with Dectin-1, the ensemble of different immune cells that express Dectin-1, and the function(s) recognized in vivo for fungal recognition. The availability of sortagged Dectin-1 mice will provide a unique opportunity to identify new targets for anti-fungal agents. PUBLIC HEALTH RELEVANCE: Our defense against fungal disease involves recognition of the invading pathogen by our immune system. The research proposed will reveal the mechanism of this detection system and the strategies used by the fungi to avoid recognition. To avoid recognition the pathogen changes or masks the identifying signature. Understanding this encounter will guide the design of new antibiotics.